Anesth Analg 2007; 105:1034-1041
© 2007 International Anesthesia Research Society
doi: 10.1213/01.ane.0000278736.81133.26
ANESTHETIC PHARMACOLOGY
The Antiproliferative Effect of Sildenafil on Pulmonary Artery Smooth Muscle Cells Is Mediated via Upregulation of Mitogen-Activated Protein Kinase Phosphatase-1 and Degradation of Extracellular Signal-Regulated Kinase 1/2 Phosphorylation
Bingbing Li, MD, PhD*,
Lingchao Yang, MD
,
Jianying Shen, MD, PhD
,
Chunshen Wang, MD
, and
Zhen Jiang, MD*
From the *Department of Anesthesiology, Zhongshan Hospital affiliated Fudan University; Department of Cardiology, School of Medicine, Xinhua Hospital affiliated Shanghai Jiaotong University; Departments of Cardiology; and Cardiac Surgery, Zhongshan Hospital affiliated Fudan University, Shanghai, China.
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Abstract
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BACKGROUND: Pulmonary hypertension is a group of diseases comprising vascular constriction and by obstructive changes of the pulmonary vasculature. Phosphodiesterase type 5 inhibitors, e.g., sildenafil, can alleviate vascular remodeling in the monocrotaline pulmonary hypertension model in rats, and inhibit the proliferation of pulmonary vascular smooth muscle cells in vitro. We examined the ability of sildenafil to inhibit platelet-derived growth factor (PDGF)-induced proliferation of porcine pulmonary artery smooth muscle cells.
METHODS: Pulmonary artery smooth muscle cell proliferation and cell cycle analysis were assessed by MTT assay and fluorescence-activated cell sorting. Western blotting was used to examine protein expression of mitogen-activated protein kinase phosphatase-1 (MKP-1) and phosphorylation level of extracellular signal-regulated kinase (ERK1/2).
RESULTS: PDGF increased cell proliferation and the percentage of cells in S phase. These effects were inhibited by pretreatment with sildenafil in a dose-dependent manner. Sildenafil (96 µM) also caused a 67% decrease in PDGF-stimulated ERK1/2 phosphorylation. Sildenafil inhibition of ERK1/2 was accompanied by a rapid induction of MKP-1. Inhibition of the cGMP-dependent kinase I 
(cGK I ) using Rp-8-BrcGMPS (25 µM) blocked sildenafil-induced MKP-1 expression. Either vanadate (12.5 µM), a phosphatase inhibitor, or Rp-8-BrcGMPS abolished the inhibitory effect of sildenafil on PDGF-stimulated phosphorylation of ERK1/2 and restored PDGF-induced cell proliferation.
CONCLUSION: This study indicates that sildenafil upregulates MKP-1 expression and promotes degradation of phosphorylation of ERK1/2, which suppresses the proliferation of pulmonary artery smooth muscle cells.
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Introduction
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Pulmonary hypertension is characterized by an increase in pulmonary vascular resistance, thrombosis formation in situ and pulmonary vascular remodeling (1,2). Accumulating evidence shows that proliferation as well as phenotype alteration of pulmonary artery smooth muscle cells play key roles in the pathogenesis and progression of pulmonary hypertension (3). Several studies indicate that over-expression of platelet-derived growth factor (PDGF), as well as PDGF-receptor mutations in the pulmonary vasculature are characteristic alterations in patients with pulmonary hypertension (4–6). Schermuly et al. reported that cultured pulmonary artery smooth muscle cells from patients with pulmonary hypertension manifested hyperplasia, and PDGF receptor antagonist STI571 reversed pulmonary vascular remodeling in two different animal models of pulmonary hypertension (7). PDGF receptors are members of the family of transmembrane tyrosine receptor kinases, autophosphorylation of which can increase kinase activity and enhance subsequent signal transduction through various pathways, including the PI3K-AKT and Raf-MEK-MAPK pathway. MAPKs phosphorylate downstream cytoskeletal proteins, other kinases, transcription factors etc., thereby orchestrating cellular proliferation, differentiation, survival, and apoptosis.
The selective phosphodiesterase type 5 inhibitor sildenafil, in addition to its vasodilation effect, prevents pulmonary vascular remodeling and right ventricular hypertrophy in the monocrotaline rat model of pulmonary hypertension (8,9). Moreover, several investigators reported inhibitory effects of sildenafil on human pulmonary artery smooth muscle cell proliferation, but underlying mechanisms remain uncertain (10,11).
Atrial natriuretic peptide, an external soluble guanylate cyclase activator, increases MAPK phosphatase-1 (MKP-1) expression in a time- and dose-dependent manner (12,13). MKP-1, a dual specificity phosphatase, is one member of the MAPK phosphatase family that functions as a negative regulator of MAPK signaling (14). Since MKP-1 can be upregulated by cGMP-increasing drugs and can repress cell growth in several types of mammalian cells via dephosphorylation of extracellular signal-regulated kinase (ERK1/2), we decided to explore the possibility that sildenafil would increase MKP-1 expression in pulmonary artery smooth muscle cells, and that this increase might play a role in the inhibitory effect of sildenafil on smooth muscle proliferation.
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METHODS
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Materials
Cell culture reagents, fetal bovine serum (FBS), and antibiotics were purchased from Gibco (NY). PDGF-BB was from Cytolab (Rehovot, Israel). Electrophoresis and protein assay reagents were from Bio-Rad (Richmond, CA). SDS/polyacrylamide gel electrophoresis and western blotting reagents were from Bio-Rad (Hercules, CA). Anti-ERK1/2 and phospho-ERK1/2 antibodies were from Cellsignaling Technology (Danvers, MA). MKP-1 antibodies were from Upstate (Lake Placid, NY). The Rp diastereomer of 8-bromoguanosine 3', 5'-cyclic monophosphothioate (Rp-8-BrcGMPS), 8-bromoguanosine 3', 5'-cyclic monophosphate (8-BrcGMP), protease inhibitors, sodium orthovanadate, and all other reagents were from Sigma Chemical (Sigma, PA). Sildenafil was kindly provided by Pfizer (Sandwich, UK).
Culture of Pulmonary Artery Smooth Muscle Cells and Treatment
After obtaining approval from the animal care and use committee at Zhongshan Hospital Fudan University, pulmonary artery smooth muscle cells were isolated by means of the explant culture method (16,17). Briefly, segments of pulmonary artery were obtained under general anesthesia from male swine (weight 20–30 kg) by careful dissection. The adventitia and intima and the outer portion of the media of each segment were carefully removed. Each segment was cut into approximately 9 mm2, placed in a culture flask and incubated at 37°C in an atmosphere of 95% air and 5% CO2. The cells that grew from the explants had become relatively confluent within a period of approximately 3 wk. They were trypsinized and further subcultured after reaching 90% confluence. A monoclonal antibody against smooth muscle -actin was used to assess the purity (>99%) of the smooth muscle cells culture (Fig. 1A and B). Unless otherwise indicated, primary cultures of pulmonary artery smooth muscle cells were maintained in RPMI-1640 containing 10% FBS and 1% antibiotic (penicillin and streptomycin mixture).
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Figure 1. Porcine pulmonary artery smooth muscle cells (PASMCs) (A) Magnification: x100 and immunofluoresence identification of smooth muscle cells -actin. (B) Magnification: x200.
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All experiments on ERK1/2 activation, MKP-1 expression, as well as cell proliferation and cell cycle assays, were performed on cells (3 days in culture) at passage 3–5. Thereafter, cells were serum-starved for 3 days in RPMI-1640 containing 0.2% FBS and 1% antibiotics. Then cells were exposed to PDGF-BB (20 ng/mL), or sildenafil at 24 µM or 96 µM followed by PDGF as indicated. In some experiments, cells were pretreated with various inhibitors for 30 min before sildenafil and subsequent exposure to PDGF, as indicated. In control groups, an equal volume of phosphate buffered saline (PBS) was substituted for the other reagents.
MTT Colorimetric Assay
The MTT test is based on the enzymatic reduction of the tetrazolium salt (MTT) in viable/metabolically active cells. Cells at approximately 90% confluency were harvested with 0.1% trypsin/0.01% edetic acid solution, seeded into a 96-well plate at a density of 2 x 104 cells per well and grown in RPMI-1640 containing 10% FBS for 3 days, followed by serum-starvation for 3 days. Cells were then incubated for 72 h with PDGF or different concentrations (24 µM, 96 µM) of sildenafil followed by PDGF with or without inhibitor pretreatment, as described above. Control cells were treated in the same way except that drug was replaced by sterile PBS. After treatment, medium was changed to fresh medium, and cells were incubated with 5 g/L of MTT for 4 h. MTT was then dissolved with 150 µL of 10% dimethyl sulfoxide for 20 min. The optical densities (OD) in the 96-well plates were determined using a microplate reader at 570 nm.
Flow Cytometry Analysis
Cells at approximately 90% confluency were harvested with 0.1% trypsin/0.01% edetic acid solution, seeded into a 6-well plate at a density of 5 x 104 cells per well and grown in RPMI-1640 containing 10% FBS for 3 days, followed by serum-starvation for 3 days. Cells were then incubated for 24 h with PDGF or different concentrations (24 µM, 96 µM) of sildenafil followed by PDGF with or without inhibitor pretreatment, as described above. Cells were rinsed with PBS, trypsinized by 0.1% trypsin/0.01% EDTA solution, and collected by centrifugation at 1000 rpm at 20°C for 5 min. The cell pellets were fixed in 70% ethanol at 4°C for at least 24 h. The fixed cells were washed twice with PBS, resuspended in PBS containing 50 g/L RNase A and 50 mg/L of propidium iodide. The suspension was incubated at 37°C for 30 min, filtered through 200 µm nylon mesh, and analyzed by flow cytometer (FACS Calibur). ModfitLT software was used for data analysis. The ratio of cells in S phase to cells in all the cycle was calculated using the formula: SPF (S Phase Fraction) = S/(G0G1 + S+ G2M) x 100%.
Immunoblot Analysis of MKP-1
Confluent serum-starved pulmonary artery smooth muscle cells were exposed to sildenafil (0–192 µM) or other treatments for 60 min. At the end of the incubation periods, lysis buffer containing protease inhibitor cocktail and phosphatase cocktail solution was added, and then cells were scraped into 1.5 mL centrifuge tubes. The cell suspension centrifuged at 12,000g for 30 min at 4°C and the protein concentration of the supernatant was determined by BCA assay. The supernatant was boiled in a loading buffer and an equal amount of proteins (18 µg protein per slot) was separated by SDS-PAGE with 12% gel. Then total protein was transferred to polyvinylidene difluoride membrane, probed with MKP-1 antibody.
Immunoblot Analysis of ERK1/2 Phosphorylation Status
Confluent, serum-starved cells were treated and protein was extracted at the indicated time as described above. Protein was examined using western blotting analysis. Briefly, equal amounts of protein (15–20 µg) were separated by SDS-PAGE, transferred to polyvinylidene difluoride membrane, probed with phospho-ERK1/2 antibody, and detected with horseradish peroxidase-conjugated secondary antibody. Phospho-ERK1/2 antibody recognizes both ERK1 and ERK2. ERK2 is the predominant isoform in pulmonary artery smooth muscle cells. Therefore, quantitation of the enhanced chemiluminescence signals was performed on ERK2. To examine the expression of ERK1/2, the membrane was washed with strip buffer in the water incubator at 50°C for 30 min, followed by blocking the membrane with 5% bovine serum albumin in PBST for 4 h. Thereafter the membrane was reprobed with specific ERK1/2 antibody.
Statistics
The results are presented as mean ± se. Analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed to compare the mean values between various groups. A P value of <0.05 was considered statistically significant.
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RESULTS
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PDGF-Mediated Pulmonary Artery Smooth Muscle Cell Proliferation and the Inhibitory Effect of Sildenafil
The percentage of cells in S phase increased dramatically from 4% to 21% after 20 ng/mL PDGF stimulation for 24 h and was accompanied by a reduction in the percentage of cells in G1 phase from 92% to 77%. These effects were inhibited by pretreatment with sildenafil at 24 µM or 96 µM (Figs. 2A and B). The OD value in MTT assay, which represents the number of viable cells or cell proliferation, markedly increased after 3 days incubation with PDGF: from 0.24 in control cells to 0.46 (P < 0.01). Sildenafil at 24 µM or 96 µM, respectively, inhibited by 22% and 28% the increase in OD value compared with PDGF-treated cells (Fig. 2C). Our FACS results indicated that the ratio of apoptotic cell is negligible among groups (Fig. 2B). Because of its close relationship with cell growth in the MAPK family, the phosphorylation status of ERK1/2 was examined. The time course of ERK1/2 phosphorylation level showed that the maximum effect occurred at 10 min. It was sustained for 1 h and returned to basal level 8 h after incubation with PDGF (Fig. 3A). Sildenafil at 24 µM or 96 µM, respectively, suppressed by 25% and 67% the phosphorylation level of ERK1/2 as compared with PDGF-treated cells (Fig. 3B).
Sildenafil-Mediated Upregulation of MKP-1 Through cGMP/cGK I Pathway
Sildenafil (0–96 µM) increased expression of MKP-1 after 1 h incubation. A further increase in concentration (to 192 µM) did not enhance MKP-1 expression, and actually reduced expression (Fig. 4A). To investigate the role of cGMP/cGK I in the sildenafil-induced upregulation of MKP-1, Rp-8-BrcGMPS at 25 µM (cGK I inhibitor) and 8-BrcGMP at 100 µM (cGMP analog) were administered alone or 30 min before sildenafil (96 µM). Rp-8-BrcGMPS alone had little effect on MKP-1 expression, but significantly reduced MKP-1 expression induced by sildenafil as compared with control cells (P < 0.01). Sildenafil (96 µM) and 8-BrcGMP (100 µM) markedly increased MKP-1 expression, compared with control cells. Exposure to 8-BrcGMP before sildenafil resulted in enhanced MKP-1 expression relative to sildenafil or 8-BrcGMP treatment alone (Fig. 4B).
The Effect of MKP-1 Inhibition on Phosphorylation Level of ERK1/2 and Cell Cycle Progression and Subsequent Pulmonary Artery Smooth Muscle Cells Proliferation
To investigate the role of MKP-1 in the suppression of pulmonary artery smooth muscle cells proliferation, we examined the phosphorylation level of ERK1/2 with different MKP-1 inhibitors. Exposure to vanadate (12.5 µM) or Rp-8-BrcGMPS (25 µM) for 30 min before sildenafil (96 µM) reversed the inhibition of ERK1/2 phosphorylation by sildenafil (Fig. 5A, lane 5–7). In contrast, the increase in phosphorylation of ERK1/2 was inhibited by sildenafil or 8-BrcGMP induced by PDGF (Fig. 5A, lane 4,5,8).
Cell cycle changes and proliferation of cells were assessed under the inhibition of MKP-1. As shown in Figures 5B and C, a 30 min preincubation with vanadate or Rp-8-BrcGMPS before exposure to sildenafil (96 µM) prevented the reduction in the percentage of cells in S phase and abolished the inhibition of cell proliferation by sildenafil; however, the compounds had no effect on cell proliferation per se. In contrast, 8-BrcGMP at 100 µM had a similar inhibitory effect as 96 µM sildenafil (26% vs 23% reduction in OD value respectively) on cell proliferation stimulated by PDGF. The magnitude of suppression by sildenafil or 8-BrcGMP of cell percentage in S phase relative to PDGF treated cells (from 21% to 11% vs 21% to 9% respectively) was also comparable. The OD value of cells only exposed to vanadate seemed to be slightly reduced compared with control cells (0.17 vs 0.21), but the difference was not statistically significant.
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DISCUSSION
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The results presented in this study show that sildenafil inhibits PDGF-induced proliferation of porcine pulmonary artery smooth muscle cells, in part by inactivating ERK1/2 via the induction of MKP-1 expression. The cGMP/cGK I signaling pathway mediates sildenafil inhibition of cell proliferation via MKP-1 expression. Inhibition of cGMP/cGK I signaling, as well as MKP-1 activity, abolishes the inhibitory effect of sildenafil on ERK activation and cell proliferation.
Previous studies demonstrated that growth factors play a significant role in the pathogenesis of pulmonary hypertension, and that proliferation or migration of pulmonary artery smooth muscle cells under aberrant growth signals accounts for the neointimal formation and medial thickening of the vascular wall (4,6,18). Our results show that activation of ERK1/2 with subsequent cell cycle progression and proliferation of pulmonary artery smooth muscle cells are enhanced after incubation with PDGF (20 ng/mL). The MAPKs family member ERK1/2 is involved in cell growth and proliferation (19,20). Consistent with other studies, the ERK1/2 signaling pathway was immediately activated and the peak of phosphorylation of ERK1/2 ensued 10 min after pretreatment with PDGF in this study. It is the phosphorylated ERK1/2 that phosphorylates downstream kinases and several immediate early gene products, which promotes cell cycle progression through the R restrict point of the G1 phase (21). The reduction in percentage of cells in G1 phase, accompanied by an increase in the number of cells in S phase in the present study, suggested that PDGF promoted G1/S transition by upregulating phosphorylation of ERK1/2. Because our data show that the level of phosphorylated ERK1/2 is sustained for only 1 h and returns to basal level 8 h later, the process of phosphorylation in the signal transudation appears to be under the precisely regulatory control to avoid over-activation of ERK1/2 phosphorylation and cell growth.
In the present study, sildenafil suppressed the proliferation of pulmonary artery smooth muscle cells. This inhibitory effect is due to inhibition of cell cycle progression and induction of G0/G1 growth arrest. Inconsistent with other investigators (11,22), sildenafil had no effect on cell apoptosis, even though relatively high doses of the drug (24, 96 µM) were used. In our pilot study, sildenafil <24 µM had little impact on cell growth, therefore increased concentrations were used in this investigation. A wide concentration range of sildenafil (10 nM–30 µM) has been used to inhibit cell proliferation in in vitro studies by different groups (10,11,23). The reason for these differences in sensitivity is unclear. It could, in part, result from the types of cell chosen.
Sildenafil, an orally active inhibitor of PDE5, increases intracellular cGMP concentration and induces a series of complex biochemical effects. Several lines of evidence indicate that cGMP-increasing agents activate downstream cGK I , which directly phosphorylates Raf-1 and uncouples the Ras and Raf interaction, thereby preventing mitogen-induced Raf-1 activation (24). In addition to interfering in MAPKs signal transduction, cGMP-increasing drugs can promote expression of cell cycle control proteins, such as p21Waf1/Cip1or p53, or downregulate expression of antiapoptotic proteins, (e.g., Bcl-2), via pathways independent of cGMP, or at least independent of cGK I activation, which suppresses vascular smooth muscle cells proliferation (25,26). However, the precise mechanism on inhibition of pulmonary artery smooth muscle cells proliferation remains uncertain.
MKP-1, a dual specificity phosphatase, can be induced by phosphorylation status of ERK and negatively regulates activity of ERK via dephosphorylation (13). Several lines of evidence show that tumor cells downregulate MKP-1 expression, and thereby gain hyperplasia characteristics. MKP-1 upregulation and stability are the underlying mechanisms of the inhibition of smooth muscle cell migration via inactivation of phosphorylation of ERK (14). Previous studies verified that MKP-1 could be induced by cGMP, which resulted in inhibition of cell migration or myocardial hypertrophy through the cGMP/cGK I pathway. However the expression of MKP-1 in porcine pulmonary artery smooth muscle cells has not been reported. Our study indicates that sildenafil upregulates MKP-1 expression in a dose-dependent manner, which can be blocked by a cGK I inhibitor and mimicked by a cGMP analog. These data indicate that the cGMP/cGK I signaling pathway is involved in the upregulation of MKP-1 by sildenafil. Subsequently, we investigated the role of MKP-1 in the inhibition of cell proliferation. Our results suggest that MKP-1 is the main factor responsible for the antiproliferative effect of sildenafil. The reasons are as follows: first, vanadate, a phosphatase inhibitor, can abolish the inhibitory effect of sildenafil on phosphorylation of ERK1/2 and cell proliferation. Second, cGK I inhibitor, which reduces MKP-1 expression, also blocks G0/G1 phase growth arrest and inhibition of proliferation and activation of ERK1/2 mediated by sildenafil. Third, cGMP analog, which increases MKP-1 expression, can mimic the antimitogenic effect of sildenafil.
Vanadate inhibits growth, when administered at larger doses, by suppressing protein synthesis; the IC50 for this effect is about 40 µM (27). In this study, the concentration used was 12.5 µM, which has been used extensively by other investigators (28–30). Although vanadate has a minor effect on phosphorylation of ERK1/2 by its dephosphorylation activity, it actually had no impact on cell cycle and growth in the present study.
In previous studies, using higher concentration of PDE5 inhibitors, cAMP content and enzyme activity of the cAMP-dependent protein kinase (PKA) could also be affected. Several lines of evidence indicate that intracellular increases in cGMP eventually cross-activate PKA, which is well known for its antimitogenic effect through phosphorylation of Raf or increased expression of p21Waf1/Cip1 (23,31,32). We cannot exclude the possibility that PKA is involved in the mechanism of inhibition of cell proliferation. Therefore, the influence of PKA on the porcine pulmonary artery smooth muscle cells deserves further investigation.
In conclusion, we report that sildenafil can suppress proliferation of pulmonary artery smooth muscle cells, in part by upregulation of MKP-1 expression and inactivation of ERK1/2 phosphorylation.
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ACKNOWLEDGMENTS
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We thank Danling Xu, Han Fu, Rongchong Huang, and Kang Yao for their support on cell culture and molecular-biological techniques.
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Footnotes
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Accepted for publication June 11, 2007.
Supported by grants from Shanghai Science and Technology Committee (No. 024119001).
Address correspondence and reprint request to Zhen Jiang, MD, Department of Anesthesiology, Zhongshan Hospital affiliated Fudan University, Shanghai, 200032, China. Address e-mail to jiangzhen66{at}yahoo.com.cn.
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REFERENCES
|
|---|
- Farber HW, Loscalzo J. Pulmonary arterial hypertension. N Engl J Med 2004;351:1655–65[Free Full Text]
- Rabinovitch M. Pathology of pulmonary hypertension. Prog Pediatr Cardiol 2001;12:223–47
- Lincoln TM, Dey N, Sellak H. Signal transduction in smooth muscle: invited review: cGMP-dependent protein kinase signaling mechanisms in smooth muscle: from the regulation of tone to gene expression. J Appl Physiol 2001;91:1421–30[Abstract/Free Full Text]
- Barst RJ. PDGF signaling in pulmonary arterial hypertension. J Clin Invest 2005;115:2691–4[Web of Science][Medline]
- Balasubramaniam V, Le Cras TD, Ivy DD, Grover TR, Kinsella JP, Abman SH. Role of platelet-derived growth factor in vascular remodeling during pulmonary hypertension in the ovine fetus. Am J Physiol Lung Cell Mol Physiol 2003;284:826–34
- Humbert M, Monti G, Fartoukh M, Magnan A, Brenot F, Rain B, Capron F, Galanaud P, Duroux P, Simonneau G, Emilie D. Platelet-derived growth factor expression in primary pulmonary hypertension: comparison of HIV seropositive and HIV seronegative patients. Eur Respir J 1998;11:554–9[Abstract]
- Schermuly RT, Dony E, Ghofrani HA, Pullamsetti S, Savai R, Roth M, Sydykov A, Lai YJ, Weissmann N, Seeger W, Grimminger F. Reversal of experimental pulmonary hypertension by PDGF inhibition. J Clin Invest 2005;115:2811–21[Web of Science][Medline]
- Schermuly RT, Kreisselmeier KP, Ghofrani HA, Yilmaz H, Butrous G, Ermert L, Ermert M, Weissmann N, Rose F, Guenther A, Walmrath D, Seeger W, Grimminger F. Chronic sildenafil treatment inhibits monocrotaline-induced pulmonary hypertension in rats. Am J Respir Crit Care Med 2004;169:39–45[Abstract/Free Full Text]
- Itoh T, Nagaya N, Fujii T, Iwase T, Nakanishi N, Hamada K, Kangawa K, Kimura H. Combination of oral sildenafil and beraprost ameliorates pulmonary hypertension in rats. Am J Respir Crit Care Med 2004;169:34–8[Abstract/Free Full Text]
- Tantini B, Manes A, Fiumana E, Pignatti C, Guarnieri C, Zannoli R, Branzi A, Galié N. Antiproliferative effect of sildenafil on human pulmonary artery smooth muscle cells. Basic Res Cardiol 2004;99:1–8[Web of Science][Medline]
- Wharton J, Strange JW, Møller GMO, Growcott EJ, Ren XH, Franklyn AP, Phillips SC, Wilkins MR. Antiproliferative effects of phosphodiesterase type 5 inhibition in human pulmonary artery cells. Am J Respir Crit Care Med 2005;172:105–13[Abstract/Free Full Text]
- Fürst R, Brueckl C, Kuebler WM, Zahler S, Krötz F, Görlach A, Vollmar AM, Kiemer AK. Atrial natriuretic peptide induces mitogen-activated protein kinase phosphatase-1 in human endothelial cells via Rac1 and NADPH oxidase/nox2-activation. Circ Res 2005;96:43–53[Abstract/Free Full Text]
- Farooq A, Zhou MM. Structure and regulation of MAPK phosphatases. Cell Signal 2004;16:769–79[Web of Science][Medline]
- Jacob A, Molkentin JD, Smolenski A, Lohmann SM, Begum N. Insulin inhibits PDGF-directed VSMC migration via NO/cGMP increase of MKP-1 and its inactivation of MAPKs. Am J Physiol Cell Physiol 2002;283:C704–13[Abstract/Free Full Text]
- Deleted in proof.
- Ross R. Growth of smooth muscle in culture and formation of elastic fibers. J Cell Biol 1971;50:172–86[Abstract/Free Full Text]
- Yasunari K, Kohno M, Balmforth A, Murakawa K, Yokokawa K, Kurihara N, Takeda T. Glucocorticoids and dopamine-1 receptors on vascular smooth muscle cells. Hypertension 1989;13:575–81[Abstract/Free Full Text]
- Merklinger SL, Jones PL, Martinez EC, Rabinovitch M. Epidermal growth factor receptor blockade mediates smooth muscle cell apoptosis and improves survival in rats with pulmonary hypertension. Circulation 2005;112:423–31[Abstract/Free Full Text]
- Lin YW, Chuang SM, Yang JL. ERK1/2 achieves sustained activation by stimulating MAPK phosphatase-1 degradation via the ubiquitin-proteasome pathway. J Biol Chem 2003;278:21534–41[Abstract/Free Full Text]
- Davis RJ. The mitogen-activated protein kinase signal transduction pathway. J Biol Chem 1993;268:14553–6[Free Full Text]
- Pagès G, Lenormand P, L’Allemain G, Chambard JC, Meloche S, Pouysségur J. Mitogen-activated protein kinases p42MAPK and p44MAPK are required for fibroblast proliferation. Proc Natl Acad Sci USA 1993;90:8319–23[Abstract/Free Full Text]
- Zhu B, Strada S, Stevens T. Cyclic GMP-specific phosphodiesterase 5 regulates growth and apoptosis in pulmonary endothelial cells. Am J Physiol Lung Cell Mol Physiol 2005;289:L196–206[Abstract/Free Full Text]
- Osinski MT, Rauch BH, Schror K. Antimitogenic actions of organic nitrates are potentiated by sildenafil and mediated via activation of protein kinase A. Mol Pharmacol 2001;59:1044–9[Abstract/Free Full Text]
- Yu SM, Hung LM, Lin CC. cGMP-elevating agents suppress proliferation of vascular smooth muscle cells by inhibiting the activation of epidermal growth factor signaling pathway. Circulation 1997;95:1269–77[Abstract/Free Full Text]
- Gu M, Lynch J, Brecher P. Nitric oxide increases p21Waf1/Cip1 expression by a cGMP-dependent pathway that includes activation of extracellular signal-regulated kinase and p70S6k. J Biol Chem 2000;275:11389–96[Abstract/Free Full Text]
- Chiche JD, Schlutsmeyer SM, Bloch DB, de la Monte SM, Roberts JD, Filippov G, Janssens SP, Rosenzweig A, Bloch KD. Adenovirus mediated gene transfer of cGMP-dependent protein kinase increases the sensitivity of cultured vascular smooth muscle cells to the antiproliferative and pro-apoptotic effects of nitric oxide/cGMP. J Biol Chem 1998;273:34263–71[Abstract/Free Full Text]
- VinÁals F, McKenzie FR, Pouysse gur J. Growth factor-stimulated protein synthesis is inhibited by sodium orthovanadate. Eur J Biochem 2001;268:2308–14[Web of Science][Medline]
- Palm-Leis A, Singh US, Herbelin BS, Olsovsky GD, Baker K, Pan J. Mitogen-activated protein kinases and mitogen-activated protein kinase phosphatases mediate the inhibitory effects of all-trans retinoic acid on the hypertrophic growth of cardiomyocytes. J Biol Chem 2004;279:54905–17[Abstract/Free Full Text]
- Xu QH, Konta T, Furusu A, Nakayama K, Lucio-Cazana J, Fine LG, Kitamura M. Transcriptional induction of mitogen-activated protein kinase phosphatase 1 by retinoids. Selective roles of nuclear receptors and contribution to the antiapoptotic effect. J Biol Chem 2002;277:41693–700[Abstract/Free Full Text]
- Englelbrecht Y, de Wet H, Horsch K, Langeveldt CR, Hough FS, Hulley PA. Glucocorticoids induce rapid up-regulation of mitogen-activated protein kinase phosphatase-1 and dephosphorylation of extracellular signal-regulated kinase and impair proliferation in human and mouse osteoblast cell lines. Endocrinology 2003;144:412–20[Abstract/Free Full Text]
- Chattergoon NN, D’Souza FM, Deng W, Chen H, Hyman AL, Kadowitz PJ, Jeter JR Jr. Antiproliferative effects of calcitonin gene-related peptide in aortic and pulmonary artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 2005;288: L202–11[Abstract/Free Full Text]
- Hayashi S, Morishita R, Matsushita H, Nakagami H, Taniyama Y, Nakamura T, Aoki M, Yamamoto K, Higaki J, Ogihara T. Cyclic AMP inhibited proliferation of human aortic vascular smooth muscle cells, accompanied by induction of p53 and p21. Hypertension 2000;35:237–46[Abstract/Free Full Text]
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Abstract
Introduction
